Sydnonimines-specific dopamine reuptake inhibitors and their use in treating dopamine related disorders

ABSTRACT

Derivatives of Sydnonimine and its analogues, which bind selectively to dopamine transporter (DAT) proteins are useful for treating and delaying the progression of disorders and illnesses that are alleviated by inhibiting dopamine reuptake.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 12/048,334, filed Mar. 14, 2008, now abandoned which claims thebenefit of U.S. Provisional Patent Application No. 60/894,739, filedMar. 14, 2007, the entire disclosures of which are incorporated byreference herein.

GOVERNMENT RIGHTS STATEMENT

The invention described herein was made with funding provided by theU.S. National Institutes of Health, under Grant Nos. IR43DA013353-01,2R44DA013353-02A1, 5R44DA013353-03, 2R44DA013353-04A1, 5R44DA013353-05and 5R44DA013353-06. The Government has certain rights in thisinvention.

FIELD OF THE INVENTION

The present invention relates to certain Sydnonimine derivatives thatbind specifically to dopamine transporter (DAT) proteins or the site ofdopamine reuptake, compositions comprising same, and the use of suchderivatives for treating, or delaying the progression of, variousdisorders and illnesses alleviated by inhibiting dopamine reuptake.

BACKGROUND OF THE INVENTION

Neurotransmitters are chemical “messengers” that function to relayelectrical signals across the gap or synaptic cleft between one neuron,or nerve cell, and another. Neurotransmitters are stored in tiny sacscalled vesicles, located at nerve endings. As an electrical signalarrives at a neuron's terminal, the vesicles move to the neural membraneand releases their neurotransmitter molecules into the synaptic cleft.The neurotransmitters formed in the pre-synaptic (or sending) neurondiffuse across the gap and lock onto binding sites or receptors on themembrane of a neighboring, post-synaptic (or receiving) neuron. Variousbiochemical processes are set into motion in the post-synaptic neuronwhen a neurotransmitter occupies a receptor on the surface thereof,including ion transport and release or inhibition of certain enzymes.The result is that a new electrical signal is generated in thepost-synaptic neuron and the signal continues on.

Dopamine is a type of neurotransmitter that is formed in the brain andeffects the processes that regulate movement, motivation, emotionalresponse and the capacity to feel pleasure and pain. Dopamine is vitalfor performing balanced and controlled movements. After dopamine becomesbound to a receptor in the process of transmitting nerve signals, it iseventually released and removed from the synaptic cleft, back into thepre-synaptic neuron or glial cell by a reuptake process which operatesunder the influence of a protein, known as dopamine transporter (DAT),present on the neuron's outer membrane. In other words, the DAT proteinacts to clear the dopamine out of the synaptic cleft, a process which isessential to normal transmission of nerve signals.

Furthermore, DAT protein is a major determinant of the intensity andduration of the dopaminergic signal. Knockout mice lacking the dopaminetransporter (DAT-KO mice) display marked changes in dopamine homeostasisthat result in elevated dopaminergic tone and pronounced locomotorhyperactivity (Gainetdinov et al. (2001) Proc. Natl. Acad. Sci.,98:11047-54; Hall et al. (2003) Neuropsychopharmacology, 28:620-8; Mateoet al. (2004) Proc. Natl. Acad. Sci., 101:372-7).

A number of behavorial disorders and other debilitating illnesses can bealleviated by therapeutic agents that bind to DAT proteins and inhibitdopamine reuptake. These include cocaine addiction, attention deficitdisorder, depression, Parkinson's disease, obesity narcolepsy, andschizophrenia, to name a few.

Cocaine addiction continues to be a major health care concern in theUnited States. According to a U.S. Department of Health and HumanServices report (aspe.hhs.gov/health/reports/cocaine/), there are over 2million cocaine users in the United States. In an October 2002 report,the Drug Abuse Warning Network (DAWN) indicated that there were 638,484emergency room (ER) visits in the U.S. in 2001 related to drug abuse,among which nearly one-third were due to cocaine.

Although research has shown that cocaine binds to variousneurotransmitters in the brain, including not only dopamine, butserotonin and norepinephrine, as well, the reinforcing effect ofcocaine, which is a factor in the addiction, is believed to be mediatedby DAT protein binding, which causes inhibition of dopamine transport.One prominent behavioral effect of cocaine and other dopamine uptakeinhibitors is the stimulation of locomotor activity. There is asignificant correlation among affinities for [3H] WIN 35,428 (a DATinhibitor) binding and potencies for stimulating activity for cocaineand structurally similar compounds compared with stimulation of mouselocomotor activity (Izenwasser et al. (2004) Eur. J. Pharmacol.,263:277-83; Kunko et al. (1998) Pharmacol. Exp. Ther., 285:277-84).There is ample evidence that attenuating dopamine receptor activity withreceptor agonists or antagonists will affect patient behavior inaddiction (Campiani et al. (2003) J. Med. Chem., 46:3822-39;Garcia-Ladona and Fox (2003) CNS Drug Rev., 9:141-58; Schlussman et al.(2003) Pharmacol. Biochem. Behav., 75:123-31; Platt et al. (2003)Psychopharmacology (Berl), 166:298-305; Vorel et al. (2002) J.Neurosci., 22:9595-603; Ellinwood et al. (2002) Eur.Neuropsychopharmacol., 12:407-15).

It has been reported that dopamine transporter-selective compounds maybe used alone or in combination with clinically available selectiveserotonin reuptake inhibitors (SSRIs) for treating cocaine abuse andaddiction (Owens et al. (2002) Encephale., 28:350-5; Zhang et al. (2002)J. Med. Chem., 45:1930-41; Sanchez et al. (2003) Psychopharmacology(Berl), 167:353-62; Fish et al. (2004) J. Pharmacol. Exp. Ther.,308:474-80). Indeed, Sora et al. demonstrated the importance of theconcurrent involvement of the dopamine transporter and serotonintransporter (SERT) proteins in the mechanism of dependency and addictionthrough evidence from neurotransmitter-transporter knockout models inmice (Sora et al. (2001) Proc. Natl. Acad. Sci., 98:5300-5). In thisstudy, it was shown, with double knockout mice models of DAT and SERT,that mice with no dopamine transporter gene and either one copy orneither copy of the serotonin transporter displayed no preference forplaces where they had previously received cocaine. That is, without theconcurrent reuptake of dopamine and serotonin, the mice are no longer“addicted” to cocaine. It is conceivable the dopamine and serotonintransport systems may have compensated for each other, and that cocainedependency and reward behavior may be mediated through this redundancyor compensatory mechanism.

Because cocaine interacts with a number of neurotransport processes inthe brain, as noted above, the discovery of a medication that is capableof antagonizing the effect of cocaine in clinical trials, withoutproducing sedative or other undesirable side effects has proven to be aformidable task, and to date not successfully accomplished. Indeed, anumber of clinically available medications approved for othertherapeutic indications, especially reuptake/transporter blockers and/orreceptor agonists, have been or are being tested in clinical trials foreffectiveness in curtailing cocaine craving, dependency, and addiction;however, none has yet demonstrated long term efficacy.

Imbalances in the dopaminergic system have been implicated ascontributing factors in the occurrence of several neuropsychiatricdisorders, including attention deficit disorder, depression and certainsymptoms of schizophrenia.

Attention deficit disorder is a learning disorder involvingdevelopmentally inappropriate inattention, with or withouthyperactivity. The primary signs of attention deficit disorder are apatient's inattention and impulsivity. Inappropriate inattention causesincreased rates of activity or reluctance to participate or respond. Apatient suffering from attention deficit disorder exhibits a consistentpattern of inattention and/or hyperactivity-impulsivity that is morefrequent and severe than is typically observed in individuals at acomparable level of development. Using positron emission tomography(PET) to study the dopamine levels in the brains of young humansubjects, it was found that lower levels of brain dopamine may be acontributing factor for ADHD children (Volkow et al., J. Neurosci. 21(2) RC 121, (2001). Methylphenidate (Ritalin®), a compound of similarpharmacological profile to cocaine, specifically increases the braindopamine level, hence exhibiting the phenotypic therapeutic effects.

The mechanism by which psychostimulants act as calming agents in thetreatment of attention-deficit hyperactivity disorder (ADHD) orhyperkinetic disorder is currently unknown. Experiments have shown thatmice lacking the gene encoding the DAT have elevated dopaminergic toneand exhibit marked hyperactive. This activity is exacerbated by exposureto a novel environment. Additionally, these mice were impaired inspatial cognitive function, and they showed a decrease in locomotion inresponse to psychostimulants. This paradoxical calming effect ofpsychostimulants depended on serotonergic neurotransmission. Theparallels between DAT knockout mice and individuals with ADHD suggestthat common mechanisms may underlie some of their behaviors andresponses to psychostimulants. Haloperidol has been shown to produce asedative effect on such mice.

Depression is one of the most common of emotional disorders, having amorbidity rate of over 10% in the general population. Depression ischaracterized by feelings of intense sadness, despair, mental slowing,loss of concentration, pessimistic worry, agitation, andself-deprecation (Harrison's Principles of Internal Medicine, 2490-2497(Fauci et al., eds., 14^(th) ed. 1998)). Depression can have physicalmanifestations including insomnia, hypersomnia, anorexia, weight loss,overeating, decreased energy, decreased libido, and disruption of normalcircadian rhythms of activity, body temperature, and endosine functions.Moreover, as many as 10% to 15% of depressed individuals displaysuicidal behavior. R. J. Bladessarini, Drugs and the Treatment ofPsychiatric Disorders: Depression and Mania, in Goodman and Gilman's ThePharmacological Basis of Therapeutics, 431 (9^(th) ed. 1996). Strategiesto increase synaptic concentrations of dopamine have been proposed asantidepressant therapies. (See e.g., D'Aquila et al., 2000, Eur. J.Pharmacol., 405: 365-373).

Schizophrenia is considered by medical professionals to be a thoughtdisorder, mood disorder and anxiety disorder. There is no known cure forschizophrenia. Thus, treatment is directed at the symptoms ofschizophrenia and often involves administration of a combination ofantipsychotic, antidepressant and antianxiety drugs. Antipsychoticdrugs, such as haloperidol, have been in use for the treatment ofschizophrenia since at least the 1950's. These established drugs act byblocking dopamine receptors and thereby control the hallucinations,delusions and confusion of schizophrenia. In the meantime, newer drugshave been introduced, e.g. quetiapine fumerate, and risperidone, whichinteract with both the dopamine and serotonin receptors, so as to treatthe broad range of schizophrenia's symptoms.

One of the principal impediments to the success of treatments forschizophrenia is that patients frequently discontinue prescribedmedication(s), especially those having undesirable side effects, such asblurred vision, dizziness, muscle spasms, cramps, tremors and otherParkinson-like symptoms.

The uncontrolled movements seen in sufferers of Parkinson's disease aredue to the degeneration of dopamine neurons, loss of nerve terminals andconsequent dopamine deficiency. Studies have shown that wild-type micetreated with a class of compounds called DAT blockers and genetic “DATknock-out” mice are both resistant to the negative effects of specificneurotoxins, such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)and 6-hydroxydopamine (6-OHDA) on dopaminergic neurons. Therefore, apharmacological blockade of DAT protein by potent and selectiveinhibitors may provide effective therapy by preventing the onset ordelaying the progression of Parkinson's disease, and offer symptomaticbenefits associated with increases in CNS dopamine levels.

Obesity is a disorder characterized by an abnormal increase of fat inthe subcutaneous connective tissues. Among the therapeutic agentscurrently used to treat obesity are those that increase food intake,such as drugs that interfere with monoamine receptors, e.g., serotoninreceptors, dopamine receptors, noradrenergic receptors and histaminereceptors.

Narcolepsy is a neurological disorder marked by a sudden recurrent,uncontrollable compulsion to sleep, also associated with cataplexy(i.e., a sudden loss of muscle tone and paralysis of voluntary musclesassociated with a strong emotion), sleep paralysis, hypnagogichallucinations and automatic behaviors. The disease afflicts all races,females and males alike. It can vary in severity, with symptoms mostcommonly appearing in a person's teens and early twenties. Narcolepsy isclinically treated using central nervous system (CNS) stimulants, suchas Ritalin® which exerts many of its effects through dopamine uptakeblockade of central adrenergic neurons, and in particular by blockingDAT proteins.

Sydnocarb (3-(1-methyl-2-phenylethyl)-N-(phenylcarbomoyl) sydnone imine)has been discovered to have a CNS stimulatory effect, marked by anincrease in locomotor activity with practically no peripheralsympathomimetic action, as described in GB Patent 1,262,830 and GermanOffenlegungsschrift 2028880. This discovery led to the synthesis ofvarious sydnocarb analogues, which also act as CNS stimulants. See, forexample, U.S. Pat. Nos. 4,277,609, 4,301,285, 4,371,697 and 4,446,322.However, sydnocarb, also known as mesocarb, and some of its closelyrelated analogues are in fact derivatives of amphetamine, a highlyaddictive psycho-stimulant. When administered to human subjects, it ishighly likely that individuals' metabolisms may convert sydnocarb backto amphetamine. Thus sydnocarb may exhibit higher abuse potential thanthose compounds that are without the propensity to be converted in thisway. Indeed, sydnocarb is listed among the prohibited stimulants in the2006 Guide to Prohibited Substances and Prohibited Methods of Doping,edition 6, Table 6, at 30, United States Anti-Doping Agency, ColoradoSprings, Colo. (December 2005) (www.usantidoping.org).

Additionally, sydnocarb and closely related derivatives are modestreuptake inhibitors. Although having some preferential affinity towardsdopamine reuptake proteins, these compounds exhibit affinity towardnorepinephrine reuptake transporters, as well.

Because of the central and peripheral role played by the DAT in thedopaminergic system, it is an attractive target for therapeuticintervention against disorders and illnesses such as those describedabove that are alleviated by compounds that bind selectively to DAT andinhibit dopamine reuptake. Compounds that inhibit specific dopaminereuptake and lack central nervous system stimulating effects areinherently more valuable medications, as such agents would likely havefewer side effects in inducing medication dependency and drug abusepotential. Accordingly, there is an ongoing interest in the developmentof such compounds.

SUMMARY OF THE INVENTION

In brief, the present invention provides, in one aspect, a method fortreatment of or delaying progression of disorders that are alleviated byinhibiting dopamine reuptake. The method involves administering to apatient in need of such treatment a therapeutically effective amount ofa compound having the formula:

wherein R₁, R₂, R₃, R₄, R₅ and R₆, independently of one another, areradicals selected from H, C₁-C₆ alkyl, OH, halogen, C₅-C₁₄ aryl, C₆-C₂₀aralkyl, C₁-C₆ alkylthio, C₁-C₆ alkoxy, SH, C₂-C₆ alkenyl, C₂-C₆alkynyl, C₃-C₆ cycloalkyl, CN, NO₂, carboxy, carbalkoxy, carboxamido,alkylsulfonyl, alkylsulfonyloxy, aminosulfinyl, monoalkylaminosulfinyl,dialkylaminosulfinyl, aminosulfonyl, monoalkylaminosulfonyl,dialkylaminosulfonyl, alkylsulfonylamino, hydroxysulfonyloxy,alkoxysulfonyloxy, alkylsulfonyloxy, hydroxysulfonyl, alkoxysulfonyl,alkylsulfonylalkyl, aminosulfonylalkyl, monoalkylaminosulfonylalkyl,dialkyaminosulfonylalkyl, aminosulfinylalkyl,monoalkylaminosulfinylalkyl, dialkylaminosulfinylalkyl;

Ra, Rb and Rc, independently of one another, represent radicals selectedfrom H, C₁-C₄ alkyl, phenyl or phenyl C₁-C₄ alkyl;

m, n and k are independent integers from 0-4, except that m+n≠0, and Rbalkyl when m+n=2; and the pharmaceutically acceptable salts of saidcompound.

According to another aspect, the present invention provides novelSydnonimine derivatives of the formula (I), above, with the proviso thatthe following known compounds are outside the scope of this aspect ofthe invention:

(i) N-phenylcarbamoyl-3-(benzyl)-sydnonimine;

(ii) N-(3′,4′-dichlorophenyl)carbamoyl-3-phenethyl-sydnonimine;

(iii) N-(p-chlorophenyl)carbamoyl-3-phenethyl-sydnonimine; and

(iv) N-(m-trifluoromethyl)carbamoyl-3-phenethyl-sydnonimine.

In a further aspect, the present invention provides pharmaceuticalcompositions comprising one or more of the sydnonimine derivativesdescribed herein in combination with a pharmaceutically acceptablecarrier medium.

The DAT inhibitor compounds described herein differ from sydnocarb, notonly in structure, but, more importantly, in their observed effects onanimal behavior. Notably, sydnocarb is a stimulant, as evidenced by itslocomotor stimulating effect in open field studies. J. Witkin et al., J.Pharm. Exptl. Therap., 288 (3):1298-1310 (1999). By contrast, the sameanimal model study, also conducted NIDA, has shown that the compounds ofthe present invention have the effect of suppressing locomotor activityin a dose dependent manner. It is expected that the central dopaminergiceffect of these compounds, without central nervous system stimulation,will lead to a myriad of therapeutic applications for which sydnocarbwould be ineffective. Moreover, unlike syndocarb and its previouslyknown analogues, the compounds of the present invention are potent andspecific dopamine reuptake inhibitors, having no appreciable affinitytowards norepinephrine reuptake proteins. Another advantage of thecompounds described herein is that they exhibit no propensity forconversion to amphetamine in vivo.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical representation of the IC₅₀/K_(i) profile ofrepresentative examples of sydnonimine derivatives used in the practiceof this invention, namely, 3-(benzyl)-sydnonimine-N-phenylcarbamoyl (a);3-(p-methyl-benzyl)-sydnonimine-N-phenylcarbamoyl (b);3-(phenylpropyl)-sydnonimine-N-phenylcarbamoyl (c); 3-(p-carboxylbenzyl)-sydnonimine N-phenylcarbamoyl) (d);3-(p-fluoro-benzyl)-sydnonimine N-phenylcarbamoyl (e);3-phenethyl-sydnonimine-N-(3′-4′-dichloro-phenyl)carbamoyl (f)′ and3-(p-nitrophenethyl)-sydnonimine-N-(3′,4′-dinitro-phenyl)carbamoyl (g).

FIGS. 2A-2D are graphs of a pharmacokinetic profile comparison showingthat the compound of Example 6 (see below; —▪—), exhibits slightlyhigher bioavailablilty than the compound of Example 3 (—♦—) at the samedosage using different routes of administration.

FIG. 3 is a graph of a pharmacokinetic profile comparison showing thatthe compound of Example 6 (—•—=10 mg/kg; 30 mg/kg) demonstrates betteroral bioavailabilty than the compound of Example 3 (—♦—=10 mg/kg; —Δ—=30mg/kg) at different dosages.

FIGS. 4A and 4B are graphs showing that the compound of Example 3demonstrates a dose dependent suppression of spontaneous locomotoractivity (NIDA).

FIGS. 5A and 5B are graphs showing that the compound of Example 6demonstrates a dose dependent suppression of spontaneous locomotoractivity (NIDA).

FIGS. 6A and 6B are graphical representations of test resultsdemonstrating that the compound of Example 3 and the compound of Example6, respectively, are capable of suppressing spontaneous locomotoractivity for at least 3 hours (—•—=DAT inhibitor and —∘—=vehicle inFIGS. 6A and 6B).

FIGS. 7A-7C are a series of graphs showing that the compound of Example3 induces significant behavioral effects in an Irwin Behavioral Battery(A: Spontaneous activity; B: Grip strength; C: Limb tone).

FIG. 8 is a graph showing that the compound of Example 3 reduceslocomotor activity in Open Field testing (—▪—=vehicle; —▾—=testcompound, 10 mg/kg; —♦—=test compound, 90 mg/kg; —▴—=buspirone, 6mg/kg).

FIGS. 9A-9D are a series of graphs showing that the compound of Example6 induces anxiolytic effects in Open Field testing (—▪—=vehicle;—Δ—=buspirone, 6 mg/kg; —▾—=test compound, 10 mg/kg; —*—=test compound,90 mg/kg).

FIG. 10 is a graph showing that the compound of Example 3 induces ananxiolytic effect in novel environment-induced feeding suppression(—▪—=vehicle; —•—=buspirone, 6 mg/kg; —▾—=test compound, 10 mg/kg;—♦—=test compound, Ex. 3, 90 mg/kg)

FIG. 11 is a graph showing that the compound of Example 3 did not affectRotarod Performance (—▪—=vehicle; —•—=EtOH; —▾—=Ex. 3, 10 mg/kg; —♦—=Ex.3, 90 mg/kg).

DETAILED DESCRIPTION OF THE INVENTION

As previously noted, the present invention includes compounds of FormulaI, above, pharmaceutical compositions comprising such compounds andmethods of using such compounds for treating various disorders andillnesses alleviated by inhibiting dopamine reuptake, or preventing ordelaying the progression of those disorders and illnesses.

It should be appreciated that compounds of Formula I, above, may haveone or more asymmetric centers and thus exist as stereoisomers,including enantiomers and diastereomers, which are usually namedaccording to the Cahn-Ingold-Prelog system. Although the structure ofFormula I is represented without regard to stereochemistry, it isintended to include all possible stereoisomers, which may be racemicmixtures or other mixtures of R and S stereoisomers (scalemic mixtureswhich are mixtures of unequal amounts of enantiomers), as well asresolved, substantially pure optically active forms, andpharmaceutically acceptable salts thereof.

Stereoisomers of the compounds of formula (I), above, can be selectivelysynthesized or separated into pure, optically-active form usingconventional procedures known to those skilled in the art of organicsynthesis. For example, mixtures of stereoisomers may be separated bystandard techniques including, but not limited to, resolution of racemicforms, normal, reverse-phase, and chiral chromatography, preferentialsalt formation, recrystallization, and the like, or by chiral synthesiseither from chiral starting materials or by deliberate synthesis oftarget chiral centers.

All of the various isomeric forms of the compound of Formula I, above,are within the scope of this invention.

As used herein, the “alkyl” refers to saturated straight and branchedchain hydrocarbon radicals, having 1-6 and preferably 1-4 carbon atoms.The term “alkenyl” is used to refer to unsaturated straight and branchedchain hydrocarbon radicals including at least one double bond, andhaving 2-7 and preferably 2-5 carbon atoms. Such alkenyl radicals may bein trans(E) or cis(Z) structural configurations. The term “alkynyl” isused herein to refer to both straight and branched unsaturatedhydrocarbon radicals including at least one triple bond and having 2-7and preferably 2-5 carbon atoms.

The term “cycloalkyl” as used herein refers to a saturated cyclichydrocarbon radical with one or more rings, having 3-14 and preferably 5or 6-10 carbon ring-atoms.

Any alkyl, alkenyl, alkynyl or cycloalkyl moiety of a compound describedherein may be substituted with one or more groups, such as halogen, OH,SH, NH₂, C₁-C₄ monoalkylamino, C₁-C₄ dialkylamino, COOH, CN, NO₂, C₁-C₄alkyl or C₁-C₄ alkoxy.

The term “aryl” as used herein refers to an aromatic hydrocarbon radicalcomposed of one or more rings and having 5 or 6-14 carbon atoms andpreferably 5 or 6-10 carbon atoms, such as phenyl, naphtnyl, biphenyl,fluorenyl, indanyl, or the like. Any aryl moiety of a compound describedherein may be substituted with one or more groups, such as halogen, OH,SH, NH₂, C₁-C₄ monoalkylamino, C₁-C₄ dialkylamino, COOH, CN, NO₂, C1-C4alkyl or C1-C4 alkoxy. The aryl moiety is preferably substituted orunsubstituted phenyl.

The term “arylalkyl” or “aralkyl” as used herein refers to radicalshaving 6 to 20 carbon atoms that combine both an aryl and an alkylgroup, as defined above. Any aralkyl moiety of a compound describedherein may optionally be substituted with one or more of the samesubstituent groups mentioned above in reference to the aryl radical.

The term “halogen” or “halo” as used herein refers to Fl, Cl, Br and I.

The term “alkoxy” refers to alkyl-O—, in which alkyl is as definedabove.

The term “alkylthio” refers to alkyl-S—, in which alkyl is as definedabove.

The term “carboxy” refers to the moiety —C(═O)OH.

The term “carbalkoxy” refers to the moiety —C(═O)O-alkyl, in which alkylis as defined above.

The term “carboxamido” refers to the moiety —C(═O)O—NR′R″, in which R′and R″, each independently represents H, alkyl, aryl or aralkyl, all aspreviously defined.

The term “alkylsulfonyl” refers to the moiety —S(═O)₂-alkyl, in whichalkyl is as previously defined.

The term “alkylsulfonyloxy” refers to the moiety —OS(═O)₂-alkyl, whereinalkyl is as previously defined.

The term “amino(monoalkylamino-, dialkylamino-)sulfinyl” refers to themoiety —S(═O)NR′R″ in which R′ and R″ each independently represents H,alkyl, aryl or aralkyl, all as previously defined.

The term “amino(monoalkylamino-, dialkylamino-)sulfonyl” refers to themoiety —S(═O)₂NR′R″, in which R′ and R″ each independently represents H,alkyl, aryl or aralkyl, all as previously defined.

The term “alkylsulfonylamino” refers to the moiety —NHS(═O)₂-alkyl, inwhich alkyl is as previously defined.

The term “hydroxysulfonyloxy” refers to the moiety —OS(═O)₂OH.

The term “alkoyxsulfonyloxy” refers to the moiety —OS(═O)₂O-alkyl, inwhich alkyl is as previously defined.

The term “alkylsulfonyloxy” refers to the moiety —OS(═O)₂-alkyl, inwhich alkyl is as previously defined.

The term “hydroxysulfonyl” refers to the moiety —S(═O)₂OH.

The term “alkoxysulfonyl” refers to the moiety —S(═O)₂O-alkyl, whereinalkyl is as previously defined.

The term “alkylsulfonylalkyl” refers to the moiety -alkyl-S(═O)₂-alkyl,wherein alkyl (each instance) is as previously defined.

The term “amino(monoalkylamino-, dialkylamino-)sulfonylalkyl” refers tothe moieties -alkyl-S(═O)₂—NR′R″, wherein alkyl is as previouslydefined, and R′ and R″ each independently represents H, alkyl, aryl oraralkyl, all as previously defined.

The term “amino(monoalkylamino-, dialkylamino-)sulfinylalkyl” refer tothe moieties -alkyl-S(═O)—NR′R″, wherein alkyl is as previously defined,and R′ and R″ each independently represents H, alkyl, aryl or aralkyl,all as previously defined.

Preferred are the compounds of Formula I, above, wherein phenyl rings Aand/or B are mono- or di-substituted. When the A and/or B ring ismono-substituted, para-substitution is preferred. When the A and/or Bring is di-substituted, 3,4di-substitution is preferred. Most preferredare compounds in which the A ring is para-substituted, e.g.,N-phenylcarbamoyl-3-(p-methyl-benzyl)sydnominine, compounds in which theB ring is 3,4-di-substituted, e.g.,N-(3′,4′-dichlorophenyl)carbamoyl3-phenethyl-sydnominine and compoundsin which the A ring is para-substituted and the B ring is3,4-di-substituted, e.g.,N-(3′,4′-dinitro-phenyl)carbamoyl-3-(p-nitrophenethyl)-sydnominine.

The term “pharmaceutically acceptable salts” as used herein refers tosalts derived from non-toxic physiologically compatible acids and bases,which may be either inorganic or organic. Thus, when a compound ofFormula I has an acid moiety, e.g., 3-(p-carboxylbenzyl),sydnominine-N-phenylcarbamoyl, useful salts may be formed fromphysiologically compatible organic and inorganic bases, including,without limitation, alkali and alkaline earth metal salts, e.g., Na, Li,K, Ca, Mg, as well as ammonium salts, and salts of organic amines, e.g.,ammonium, trimethylammonium, diethylammonium, andtris-(hydroxymethyl)methylammonium salts. The compounds of the inventionalso form salts with organic and inorganic acids, including, withoutlimitation, acetic, ascorbic, lactic, citric, tartaric, succinic,fumaric, maleic, malonic, mandelic, malic, phthalic, salicyclic,hydrochloric, hydrobromic, phosphoric, nitric, sulfuric, methanesulfonic, naphthalene sulfonic, benzene sulfonic, toluene sulfonic andsimilar known, physiologically compatible acids. In addition, when acompound of Formula I contains both a basic moiety and an acidic moiety,zwitterions (“inner salts”) may be formed and are included within theterm “salt(s)” as used herein.

The sydnominine derivatives described herein, including thepharmaceutically acceptable salts thereof, can be conveniently preparedby those having ordinary skill and experience in organic synthesis,using known starting materials, and following the general syntheticscheme shown below, in which the radicals R₁-R₆ are as previouslydefined:

Chemical reactions for the preparation of specific sydnomininederivatives which may be used in the practice of this invention aredescribed in further detail hereinbelow. The starting materials forthese reactions are available from commercial sources. See also, U.S.Pat. No. 3,277,108.

In vitro studies have been performed that demonstrate the specific DATinhibition activity of the compounds of the invention. DAT inhibitionactivity was tested according to the procedure described by J. Javitz etal., Mol. Pharmacol., 26: 35-44 (1984). The test results for a number ofrepresentative compounds of the invention are reported hereinbelow.

As used herein, the expression “method of treating disease alleviated byinhibiting DAT” refers to a treatment using one or more of the compoundsdescribed above, which provides relief either by freeing the recipientof a disease or condition mediated by DAT or easing the symptoms oreffects of such disease or condition. The method of the invention isintended for treating, preventing, managing and/or delaying theprogression of the following: pulmonary conditions such as lung edema;ischemia-reperfusion injury; cardiac conditions, such as acutedecompensated heart failure and the cardiorenal syndrome;hyperprolactinaemia (BrE), hyperprolactinemia (AmE) andmicroprolactinoma; pain including chronic or neuropathic pain;catatonic, dyskinesia, restless legs syndrome and related movementdisorders; stress, chronic posttraumatic stress disorder, anxietydisorders, obsessive-compulsive disorders, postpartum depression;schizophrenia, manic, bipolar, and affective disorder; executivefunction disorders, such as ADHD, Tourette syndrome and autism; cocaine,amphetamine, alcohol dependency, and addictive behavior, such aspathological gambling; neuroendocrinal regulatory disorders;inflammatory conditions, autoimmune diseases and rheumatism; neoplasticdisorders, such as pituitary carcinomas, macroprolactinomas; visualsensory disorders, color deficiency; and ejaculatory and related sexualdysfunction. The diseases and conditions enumerated above are given byway of example and not by way of limitation.

In general, the compounds of the invention can be administered toachieve specific dopamine reuptake inhibition by using any acceptablemeans known in the art, either alone or in combination with one or moreother therapeutic agent. Thus, the active agent(s) can be administeredenterally, parenterally, such as by intravenous infusion, intramuscular,intraperitoneal or subcutaneous injection, by liposome-mediateddelivery, vaginally, by inhalation or insufflation, transdermally or byotic delivery.

Normally, a daily dose of the compound of the invention in the rangefrom about 0.01 mg to about 200 mg/kg of body weight can beadministered. A daily dose of from 0.1 to 100, and preferably from 1 to30 mg/kg per day in one or more applications per day should be effectiveto produce the desired result. By way of example, a suitable dose fororal administration would be in the range of 1-30 mg/kg of body weightper day, whereas a typical dose for intravenous administration would bein the range of 1-10 mg/kg of body weight per day. Of course, as thoseskilled in the art will appreciate, the dosage actually administeredwill depend upon the condition being treated, the gender, age, healthand weight of the recipient, the type of concurrent treatment, if any,and the frequency of treatment. Moreover, the effective dosage amountmay be determined by one skilled in the art on the basis of routineempirical activity testing to measure the bioactivity of the compound(s)in a bioassay, and thus establish the appropriate dosage to beadministered.

The compounds of the invention will typically be administered from 1-4times a day, so as to deliver the above-mentioned daily dosage. However,the exact regimen for administration of the compounds and compositionsdescribed herein will necessarily be dependent on the needs of theindividual subject being treated, the type of treatment administered andthe judgment of the attending medical specialist. As used herein, theterm “subject” includes both humans and animals.

The compounds of the invention may be administered as such, or in a formfrom which the active agent can be derived, such as a prodrug. A prodrugis a derivative of a compound described herein, the pharmacologic actionof which results from the conversion by chemical or metabolic processesin vivo to the active compound. Prodrugs include, without limitation,ester derivatives of the compounds of formula I, above. Other prodrugsmay be prepared according to procedures well known in the field ofmedicinal chemistry and pharmaceutical formulation science. See, e.g.,Lombaert et al., J. Med. Chem., 37: 498-511 (1994); and Vepsalainen,Tet. Letters, 40: 8491-8493 (1999).

The DAT specific compounds described herein and the pharmaceuticallyacceptable salts thereof are preferably formulated in unit dosage formfor ease of administration and uniformity of dosage. The expression“unit dosage form” as used herein refers to a physically discrete unitof the active agent appropriate for the subject to be treated. Each doseshould contain the quantity of active ingredient calculated to producethe desired therapeutic effect, either as such, or in association withthe selected pharmaceutical carrier medium and/or supplemental activeagent(s), if any. Typically, the DAT inhibitory compounds of theinvention will be administered in dosage form containing from about 0.01mg to about 200 mg of the active ingredient, with a range of about 30 mgto about 100 mg being preferred.

The orally administered dosage unit may be in the form of tablets,caplets, dragees, pills, semisolids, soft or hard gelatin capsules,aqueous or oily solutions, emulsions, suspensions or syrups. Suitabledosage forms for parenteral administration include injectable solutionsor suspensions, suppositories, powder formulations, such asmicrocrystals or aerosol spray. The active agent may also beincorporated into a conventional transdermal delivery system.

A pharmaceutical composition in accordance with the present inventioncomprises one or more of the compounds of Formula I, above, incombination or admixture with a pharmaceutically acceptable carriermedium. The composition may, if desired, be administered in conjunctionwith one or more supplemental active agents. For example, the DATinhibiting agent may be used in combination with L-dopa for thetreatment of Parkinson's disease; or in combination with a selectiveserotonin reuptake inhibitor (SSRI) for the treatment of depression andor cocaine abuse and addiction; or in combination with dopamine D2antagonist for the treatment of schizophrenia; or in combination withcholinergic modulators for the treatment of Alzheimer disease or otherdiseases or conditions in which patients have a cognitive deficit. Thecompound(s) of the invention may be administered either simultaneously(e.g., in the same formulation or not) or sequentially with thesupplemental therapeutic agent(s).

As used herein, the expression “pharmaceutically acceptable carriermedium” includes any and all solvents, diluents, or other liquidvehicle, dispersion or suspension aids, surface agent agents, isotonicagents, thickening or emulsifying agents, preservatives, solid binders,lubricants, fillers and the like as suited for the particular dosageform desired. Remington: The Science and Practice of Pharmacy, 20^(th)edition, A. R. Genaro et al., Part 5, Pharmaceutical Manufacturing, pp.669-1015 (Lippincott Williams & Wilkins, Baltimore, Md./Philadelphia,Pa. (2000)) discloses various carriers used in formulatingpharmaceutical compositions and known techniques for the preparationthereof. Except insofar as any conventional pharmaceutical carriermedium is incompatible with the DAT inhibitor compounds of the presentinvention, such as by producing an undesirable biological effect orotherwise interacting in an deleterious manner with any othercomponent(s) of a formulation comprising such compounds, its use iscontemplated to be within the scope of this invention.

For the production of solid dosage forms, including hard and softcapsules, the therapeutic agent may be mixed with pharmaceuticallyinert, inorganic or organic excipients, such as lactose, sucrose,glucose, gelatine, malt, silica gel, starch or derivatives thereof,talc, stearic acid or its salts, dried skim milk, vegetable, petroleum,animal or synthetic oils, wax, fat, polyols, and the like. For theproduction of liquid solutions, emulsions or suspensions or syrups onemay use excipients such as water, alcohols, aqueous saline, aqueousdextrose, polyols, glycerine, lipids, phospholipids, cyclodextrins,vegetable, petroleum, animal or synthetic oils. For suppositories onemay use excipients, such as vegetable, petroleum, animal or syntheticoils, wax, fat and polyols. For aerosol formulations, one may usecompressed gases suitable for this purpose, such as oxygen, nitrogen andcarbon dioxide. The pharmaceutical composition or formulation may alsocontain one or more additives including, without limitation,preservatives, stabilizers, e.g., UV stabilizers, emulsifiers,sweeteners, salts to adjust the osmotic pressure, buffers, coatingmaterials and antioxidants.

The present invention further provides controlled-release orsustained-release therapeutic dosage forms for the pharmaceuticalcomposition, in which the composition is incorporated into a deliverysystem. This dosage form controls release of the active agent(s) in sucha manner that an effective concentration of the active agent(s) in thebloodstream can be maintained over an extended period of time, with theconcentration in the blood remaining relatively constant, to improvetherapeutic results and/or minimize side effects. Additionally, acontrolled-release system would provide minimum peak to troughfluctuations in blood plasma levels of the active agent.

In the pharmaceutical compositions of the invention, the active agent(s)may be present in an amount of at least 0.5 and generally not more than95% by weight, based on the total weight of the composition, includingcarrier medium and/or supplemental active agent(s), if any. Preferably,the proportion of active agent(s) varies between 30-90% by weight of thecomposition.

While not wishing to be confined to any particular theory as to thebiochemical mechanism of action of the compounds described herein,considering that these compounds have shown 1) highly potent andspecific interaction with dopamine reuptake proteins, 2) fast onsiteabsorption and distribution (e.g. brain), and 3) little interaction withother proteins including the most prevalent metabolic enzymes, it isbelieved that these compounds may be used 1) alone to ameliorate diseaseconditions, central and or peripheral, when endogenous dopaminergicfunctions are augmented by the inhibition of dopamine reuptake; 2) incombination with dopamine (or dopaminergic agonists) to providesynergistic effects of augmented endogenous dopaminergic functions anddrug effects; and 3) in combination with other than dopaminergicmechanism medications, when the treatment of disease requires theconsideration of complex and multifaceted disease etiology.

The following examples are provided to describe the invention in furtherdetail. These examples are provided for illustrative purposes only andare not intended to limit the invention in any way.

EXAMPLE 1 Preparation of N-phenylcarbamoyl-3-(benzyl)-sydonimine

1.2 ml 7.5N aq. HCl was stirred (at 0° C.) into a mixture of 0.94 gbenzyl amine and 0.58 g of potassium cyanide in 2 ml of water. 0.7 gformaldehyde was then added dropwise into the mixture. The resultingmixture was stirred at room temperature for 2 hours and then cooled to0° C. A solution of 0.62 g sodium nitrite in 1 ml water was added slowlydropwise to the mixture followed by the addition of 1.2 ml 7.5N HCl aq.solution while cooling. The mixture was stirred at room temperature for1 hour. Ether was used to extract the resulting mixture three times. Thecombined ether solution was dried with anhydrous sodium sulfate. Thesolvent was removed under reduced pressure.N-nitroso-benzylaminoacetonitrile (see reaction scheme) was obtained asa yellow oil. The nitroso intermediate was then treated with 500 ml HClethereal solution (2.0 M) and stirred for 30 minutes at roomtemperature. White precipitate was obtained and recrystalized with2-propanol, resulting in a white crystal. 2.84 g of the3-benzyl-sydnonimine hydrochloride thus produced was dissolved in 25 mlof water. To the solution was added 1.34 g sodium bicarbonate at 0° C.2.45 ml of phenyl isocyanate was then added dropwise to the mixture andstirred at 0° C. for 4 hours to the resulting mixture was added 10 ml ofether to help the yellow crystal precipitate. Methanol was used assolvent for recrystalization. The desired product was obtained as yellowcrystal.

EXAMPLES 2-7

The compounds N-phenylcarbamoyl-3-(p-carboxylbenzyl-syndonimine (2);N-phenylcarbamoyl-3-(p-methyl-benzyl)-sydnonimine (3);N-(3′,4′-dichlorophenyl) carbamoyl-3-phenethyl sydnonimine (4);N-(3′,4′-dinitrophenyl) carbamoyl-3-p-nitrophenethyl sydnonimine (5);N-phenylcarbamoyl-3-(phenylpropyl)-sydnonimine (6); andN-phenylcarbamoyl-3-(p-fluoro-benzyl)-sydnonimine (7) were synthesizedfollowing essentially the same procedure as described in Example 1,above, with appropriate substitution of different starting materials inequivalent amounts.

EXAMPLE 8 I. Dopamine Transporter Binding Assay [3H]WIN 35,428

This assay was carried out according to the procedure described byJavitch, J. J. et al., Mol. Pharmacol. 26: 35-44; 1984

A. Tissue Preparation (Prepare All Solutions On Ice)

1. Thaw frozen brains from male Guinea Pigs (in assay buffer) and placein 50 mM TRIS-HCl (pH 7.4 at 25° C. with 120 mM NaCl). Isolate striatum.

2. Use a Polytron to homogenize tissue in 20 vols. (w/v) of 50 mMTris-HCl (pH 7.4 at 25° C. with 120 mM NaCl).

3. Centrifuge homogenate at 48,400×g for approximately 10 minutes at 4°C. Discard supernatant.

4. Wash pellet an additional time as described in steps 2 and 3.

5. Store pellet on ice until needed for binding assay.

6. Using a Polytron (setting 5; approximately 10 seconds) resuspendpellet in 50 mM Tris-HCl (pH 7.4 at 25° C. with 120 mM NaCl) to aninitial concentration of 10 mg/ml (original wet weight), such that thefinal concentration is 8 mg/ml or 4.0 mg tissue/tube.

B. Binding Reaction

1. Each tube receives the following components:

-   -   50 ul drug or vehicle    -   50 ul [3H]-WIN 35,428    -   400 ul tissue suspension.

2. Initiate binding reaction with the addition of tissue, and incubatefor 120 minutes at 0° C. (on ice).

3. Terminate binding reaction by rapid filtration of tube contents ontountreated Whatman GF/C filters.

4. Rinse the assay tubes once with ice cold 50 mM TRIS HCl (pH 7.4 at25° C. with 120 mM NaCl, BSA), then rapidly rinse the filters with 6×1mls/tube of the same wash buffer.

5. Radioactivity trapped onto the filters is assessed using LSM machine.Let sit for 3 hours in scint cocktail.

C. Dopamine Tranasporter Binding Assay Materials And Reagents

1. [3]H-WIN 35-428 is diluted to a concentration of 60 nM in 50 mM TRISHCl (pH 7.4 at 25° C. with 120 mM NaCl). Thus, the final ligandconcentration is 6 nM.

2. Non-specific binding is defined as that remaining in the presence of1×10-6 M GBR12909 (room temperature cabinet). GBR12909 MW=523.5 g/mol.Will solubilize in 4% DMSO (sonicate 15 minutes). Aliquots can be used(1E-3).

3. The reference compound for the assay is GBR12909 and is diluted in 4%DMSO and is then run at the following final concentrations: 1×10⁻¹⁰,5×10⁻¹⁰, 2×10⁻⁹, 5×10⁻⁹, 1×10⁻⁸, 2×10⁻⁸, 5×10⁻⁸, 2×10⁻⁷, 5×10⁻⁷, 1×10⁻⁶,5×10⁻⁶.

4. The positive control GBR12909 and is run at the final concentrationsof 2×10-8, 1×10⁻⁷, 5×10⁻⁷M.

5. The K_(d) for the receptor is 28.0 nM.

Buffers

Assay Buffer 1 L 2 L 3 L 4 L 50 mM Tris- 6.06 g 12.12 g 18.18 g 24.24  HCl pH 7.4 120 mM NaCl 7.01 g 14.02 g 21.03 g 28.04 gWash Buffer (Add 1 g BSA/1 L Assay Buffer)

50 mM Tris-HCl pH 7.4 24.2 g/4 L 120 mM NaCl 28.0 g/4 L BSA   4 g/4 LKi: 2.25-22.5 e-9

II: Norepinephrine Transporter (Human Recombinant) Binding Assay

This assay was carried out in accordance with the procedure described byRaisman, R, et al. Eur. Jrnl. Pharmacol. 78: 345-351 (1982) withmodifications. See also, Langer, S., et al., Eur. Jrnl. Pharmacol, 72:423-4 (1981).

Tissue Preparation

1. hrNET (Receptor Biology; RBNET) is diluted in assay buffer to 2.5microunits/ml, so that each tube receives 0.5 microunits, or 2microunits/ml.

NOTE: 100 microassays, add 20 mls of buffer.

Binding Reaction

1. Each tube receives the following components:

-   -   25 ul drug or vehicle    -   25 ul [³H]-Nisoxetine    -   200 ul tissue suspension.

2. Initiate binding reaction with the addition of tissue, and incubateat room temperature for 1 hour.

3. Terminate binding reaction by rapid filtration of tube contents onto0.1% PEI treated GF/C filters (TopCount).

4. Rinse the assay tubes once with ice cold 50 mM NaCl, then rapidlyrinse the filters with 6×1 ml/tube of the same wash buffer.

5. Radioactivity trapped onto the filters is assessed using liquidscintillation spectrophotometer after soaking the filters for at least 1hour in scintillation cocktail.

Materials And Reagents

1. The receptor source is human recombinant/CHO.

2. [³H]-Nisoxetine, diluted to a concentration of 10 nM in assay buffer,is used as the radioligand. Thus, the final ligand concentration is 1nM.

3. Non specific binding is defined as that remaining in the presence of1×10⁻⁶M desipramine (MW=302.8) (Make fresh: in bag with dessicatingrocks 1, dissolves in water 1E-3).

4. The reference compound for the assay is desipramine. Desimpramine isrun at following final concentrations:

-   -   1×10⁻¹⁰, ²×10⁻¹⁰, 5×10⁻¹⁰, 1×10⁻⁹, 2×10⁻⁹, 5×10⁻⁹, 1×10⁻⁸,        2×10⁻⁸, 5×10⁻⁸, 1×10⁻⁷, 2×10⁻⁷, 5×10⁻⁷M

5. The positive control is any of the above run at the finalconcentrations of:

-   -   1×10⁻⁹, 5×10⁻⁹, 2×10⁻⁸ M.

6. The K_(d) for the receptor is 3 nM.

Buffers

MW (g/mole) g/1 L Incubation Buffer: 50 mM Tris, pH 7.4 121 6.06 5 mMKCl 74.6 0.38 120 mM NaCl 58.4 7.02 Wash Buffer: 50 mM NaCl 58.4 3.0

The results of the DAT and NET binding assays are set forth in thefollowing table:

INH at 10 INH at 10 Ex. No. Name Structure μM DAT μM NET Ki DAT 13-(benzyl)- syndonimine-N- phenylcarbamoyl

90% 0   36 nM 2 3-(p-carboxylbenzyl)- syndonimine N- phenylcarbamoyl

92%   11%  2.7 nM 3 3-(p-methyl-benzyl)- syndonimine-N- phenylcarbamoyl

94%   14% 0.19 nM 4 3-phenethyl- syndonimine-N- (3′,4′-dichlorophenyl)carbamoyl

99%   2%   40 nM 5 3-(p-nitrophenethyl) syndonimine-N-(3′,4′-dinitrophenyl) carbamoyl

85%   4%  100 nM 6 3-(phenylpropyl)- syndonimine-N- phenylcarbamoyl

96% 1.44%   25 nM 7 3-(p-fluorobenzyl)- syndonimine-N- phenylcarbamoyl

99%   15% 0.46 nM

Preferred compounds of the invention have a DAT binding inhibition (at10 μm) of greater than or equal to 90% and a NET binding inhibition (at10 μM) of less than or equal to 15%; or a ratio of DAT:NET bindinginhibition (at 10 μM) of at least 9:1.

EXAMPLE 9

The following materials and methods are provided to facilitate thepractice of Example 9, in which drug concentration in certain biologicalsamples were assessed using different routes of administration.

Parameters Used In Dosing And Drug Administration of the Compound ofExample 3

Dose route Dose range Dose volume (Use a 1-ml syringe with appropriateoral feeding needle.) i.v. 10 mg/kg 5-10 × bodyweight   (5-10 ml/kg,i.e. a 25 g mouse receives 125-250 ul) i.p 10 mg/kg 10 × bodyweight Oral10.30 mg/kg 10 × bodyweight

Mouse Strain

Adult C57BL/6 mice (preferred 16 weeks old).

Time Point

Pre-dose, 5, 15, 30, 60, 120, 240 and 480 mins for IV.

Pre-dose, 15, 30, 60, 120, 240, 480 and 1,440 mins for IP and oral.

Three mice per time point. If all three routes are conducted in sameday, one group of pre-dose is efficient.

Dosing Formula

10% EtOH

0.05-0.08% 7.5N HCL

30% Captisol in Saline

Final PH at 2.5-3

EXAMPLE 10 Spontaneous Locomotor Activity (Open Field Study)

A dose response study was conducted of locomotor depression induced bythe compound of Example 3, according to NIDA's Medication DevelopmentDivision (MDD) locomoter activity studies standard protocol. The studywas conducted using 40 Digiscan locomotor activity testing chambers(40.5×40.5×30.5 cm) housed in sets of two, within sound attenuatingchambers. A panel of infrared beams (16 beams) and correspondingphotodetectors were located in the horizontal direction along the sidesof each activity chamber. A 7.5 W incandescent light above each chamberprovided dim illumination. Fans provided an 80 dB ambient noise levelwithin the chamber. Separate groups of 8 non-habituated male SwissWebster mice (Hsd:ND4, aged 2-3 months) were injected via theintraperitoneal (IP) route with either vehicle (2% methyl cellulose) orthe compound of Example 3, above, (3, 10, 30 or 100 mg/kg) twentyminutes prior to locomotor activity testing. Just piro to placement inthe apparatus, all mice received a saline injection IP. In all studies,horizontal activity (interruption of photocell beams) was measured for 1hour within 10 minute periods. Testing was conducted with one mouse peractivity chamber.

The same protocol was followed in a study of locomotor depressioninduced by the compound of Example 6.

EXAMPLE 11 Time-Course (8 hr.) Mouse Locomotor Activity Testing

A time course/dose response study of the locomotor depression inducingeffect of the compound of Example 3 was conducted, according to the sameMDD locomotor activity studies time course protocol describedimmediately above, except that groups of 8 mice were injected witheither vehicle (2% methylcellulose) or the compound of Example 3 (3, 10,30 or 100 mg/kg), immediately prior to locomotor activity testing.Behavioral observations were recorded on each mouse at 30, 120 and 480minutes following 100 mg/kg of the test compound. The vehicle used inthis study was 2% methylcellulose.

A separate time course/dose response study of the locomotor depressioninducing effect of the compound of Example 6 was also conducted, underthe same conditions described above for testing of the compound ofExample 3. Additional groups of 8 mice were injected with either vehicle(2% methylcellulose) or the test compound (3, 10, 30 or 100 mg/kg),immediately prior to locomotor activity testing. Behavioral observationswere recorded on each mouse at 30, 120 and 480 minutes following 100mg/kg of the test compound. The vehicle used in this study was 2%methylcellulose.

Results

The inventors have identified DAT selective inhibitors for use in thetreatment and prevention of cocaine abuse and other disorders associatedwith aberrant dopamine reuptake. Two such inhibitors, namely, thecompounds of Examples 3 and 6, above, are well tolerated up to at least200 mg/kg. Observable changes in behavior occurred in minutes and lastedfor about 2-3 hours. Notably subjects treated with the compound ofExample 3 became lethargic at dosages above 125 kg/mg, whereas thiseffect was reduced at higher concentrations of the compound of Example6.

In the studies using adult C57BL/6 mice (preferably approximately 16weeks old), dosing and drug administration parameters for the compoundof Example 3 were explored. Table 2 summarizes the results of thesestudies. Clearly, the compound is suitable for oral administration andlasts in the plasma up to 2-3 hours. Moreover, the compound of Example 3reaches the brain whether administered orally or via intravenousadministration. See Table 3.

TABLE 2 Plasma Concentration (ng/mL) by Different Routes of DrugAdministration Time 10 mg/kg (IV) 10 mg/kg (IP) 10 mg/kg (PO) 30 mg/kg(PO) 0 18.33 21.00 10.60 8.93 5 2091.03 973.00 218.50 837.87 15 2110.87315.97 115.45 669.80 30 915.00 544.25 59.90 374.53 60 416.63 115.4021.30 113.87 120 139.27 82.47 17.57 41.87 240 47.43 34.03 15.80 48.53480 94.63 31.20 14.30 13.40

TABLE 3 Brain Drug Concentration (ng/mL) by Different Routes of DrugAdministration Time 10 mg/kg (IV) 10 mg/kg (IP) 10 mg/kg (PO) 30 mg/kg(PO) 0 95.10 0.00 107.55 123.97 5 2313.10 1456.65 417.80 868.87 151723.27 374.43 297.60 844.87 30 1139.30 242.00 149.93 626.63 60 464.40189.93 158.00 217.60 120 78.70 70.20 165.13 203.07 240 177.87 143.1397.47 183.03 480 48.95 122.95 163.00 168.35Additional pharmacokinetic studies were performed, as shown in FIGS.2A-2D. It appears that the compound of Example 6 exhibits slightlybetter bioavailability in the plasma and brain when compared to thecompound of Example 3 at the same dosage. The compound of Example 6 alsoexhibits relatively higher oral bioavailability when administered atdifferent dosages. See FIG. 3.

The spontaneous locomotor activity assessments were performed followingadministration of the DAT inhibitors of the present invention accordingto the procedure described in Tella et al. (1996) Pharmacol Biochem.Behav. 54:343-54; Elmer et al. (1996) Pharmacol Biochem Behav53:911-918. The results of these assessments appear in FIGS. 4 and 5.FIGS. 4A and 4B are graphs that show that the compound of Example 3demonstrates a dose dependent suppression of spontaneous locomotoractivity. The compound of Example 6 showed a similar dose dependentresponse, as can be seen in FIGS. 5A and 5B.

The results of the above-described time-course mouse locomotor activityexperiment using the compound of Example 3 are set forth in FIG. 6A,which shows average horizontal activity counts/10 min as a function oftime (0-8 hr) and dose of the compound (top to bottom panels). Treatmentwith this compound resulted in time- and dose-dependent depression oflocomotor activity following 30 and 100 mg/kg. Depressant effects of 30and 100 mg/kg occurred within 10 minutes following injection and lasted140 to 160 minutes. The period 20-50 min was selected for analysis ofdose-response data because this was the time period in which maximalsuppression first appeared as a function of dose. The mean averagehorizontal activity counts/10 min for this 30-min period were fit to alinear function of log₁₀ dose of the descending portion of thedose-effect curve (3 to 100 mg/kg dose range). The ID₅₀ (dose producing½ maximal depressant activity, where maximal depression=0 counts/30mint) was estimated to be 24.0 mg/kg.

A two-way analysis of variance conducted on horizontal activitycounts/10 min indicated a significant effect for Treatment F (4,35)=5.35, p=0.002, 10-Minute Periods F (47, 1645)=42.63, p<0.001, andfor the interaction of Periods and Treatment F (188, 1645)=2.26,p<0.001. A one-way analysis of variance conducted on log₁₀ horizontalactivity counts for the 20-50 min time period (maximal depressanteffect) indicated a significant effect of Treatment F (4, 35)-27.59,p<0.001, and planned comparisons (a priori contrast) against the vehiclegroup showed a significant depressant effect for 30 and 100 mg/kg(ps<0.05 denoted on FIG. 6A with an asterisk).

The results of the time-course mouse locomotor activity experiment usingthe compound of Example 6, is provided in FIG. 6B, which shows averagehorizontal activity counts/10 min as a function of time (0-8 hr) anddose of the test compound (top to bottom panels). Treatment with thiscompound resulted in time-dependent depression of locomotor activityfollowing 100 mg/kg. Depressant effects of 100 mg/kg occurred within 10minutes following injection and lasted 160 minutes. The period 50-80 minwas selected for analysis of dose-response data because this was thetime period in which maximal suppression first appeared as a function ofdose. The mean average horizontal activity counts/10 min for this 30-minperiod were fit to a linear function of log₁₀ dose of the descendingportion of the dose-effect curve (10 to 100 mg/kg dose range). The ID₅₀(dose producing ½ maximal depressant activity, where maximaldepression=0 counts/30 min) was estimated to be 30.2 mg/kg.

A two-way analysis of variance conducted on horizontal activitycounts/10 min indicated a significant effect of Treatment F (4,35)=6.84, p<0.001, 10-Minute Periods F(47, 1645)=48.92, p<0.001, and theinteraction of Periods and Treatment F(188, 1645)=1.64, p<0.001. Aone-way analysis of variance conducted on log 10 horizontal activitycounts for the 50-80 min time period (maximal depressant effect)indicated a significant effect of Treatment F (4, 35)=6.01, p=0.001, andplanned comparisons (a priori contrast) against the vehicle group showeda significant depressant effect for 100 mg/kg (ps<0.05 denoted on FIG.6B with an asterisk).

To further characterize the pharmacological effects of these compounds,Irwin behavioral battery testing was performed.

Testing occurred towards the end of the light cycle between 8-11 a.m.(lights go off at 11:00 a.m.). The mice were acclimated to the testingroom for at least 30 minutes. The Irwin test equipment (timer, viewingjar and support, open arena, grid, ruler, sound box, wood stocks,kimwipes box) was placed under a laminar flow hood. The mouse was firstplaced in the viewing jar for five (5) minutes and the followingparameters were scored:

1. General appearance—coat quality, whiskers

2. Body position

3. Spontaneous activity

4. Respiration rate

5. Tremors

6. Twitches

7. Bizarre behavior—comments

8. Convulsions

9. Defecation—number of feces at the end of the session

At the end of the 5-minute period, the viewing jar with the muose in itwas transferred to the arena, where the jar was disassembled and themouse released into the arena. The mouse was not handled by theexperimenter. The following parameters were scored in the arena in thefollowing order:

1. Transfer arousal—during the 10 first seconds

2. Locomotor activity—number of squares entered by all four feet in 30seconds

3. Palpebral closure

4. Piloerection

5. Startle response—90 dB sound from clickbox 30 cm above arena

6. Gait

7. Pelvic elevation

8. Tail elevation—during forward motion

9. Touch escape—finger stroke from above

10. Tail pinch—with forceps 3 cm from the base of tail

11. Body temperature—hypo- or hyperthermia

12. Positional passivity—struggle response to sequential handling bytail, neck, hind legs, or held supine

Then the mouse was picked up by its tail and kept above the arena toscore the following set of parameters:

1. Trunk curl

2. Limb grasping

3. Visual placing—extension of forelimbs when animal is lowered by baseof tail from a height of 15 cm above a wire grid

4. Grip strength—mouse is lowered and allowed to grip the grid; gentlehorizontal backwards pull is applied

5. Body tone—sides of mouse are compressed between thumb and indexfinger

6. Pinna reflex—mouse is gently restrained on grid and the proximal partof the inner cathus is touched lightly with the tip of fine wire probe;ear retraction is observed

7. Corneal reflex—mouse is gently restrained no grid and the cornea istouched lightly with the side of fine wire probe; eye-blink response isobserved

8. Toe pinch—the mid digit of hind foot is gently compressed laterallywith fine forceps. The hind limbs are lifted clear of the grid

9. Wire Maneuver—mouse is held above the wire by tail suspension andlowered to allow the forelimbs to grip the horizontal wire; the mouse isheld in extension and rotated around to the horizontal and released

The mouse was then placed in supine restraint to score the belowparameters in the following order:

1. Body length—from tip of nose to base of tail (mm)

2. Skin color—plantar surface and digits of forelimbs

3. Heart rate—felt by palpation below sternum

4. Limb tone—resistance to gentle finger tip pressure on plantar surfaceof left/right hind paw

6. Lacrimation

7. Pupil reflex

8. Salivation

9. Provoked biting—dowel rod gently inserted between the teeth at theside of the animal's mouth

10. Irritability

The final three parameters were scored with the mouse above or in thearena:

1. Righting reflex—mouse is held by the tail and flicked backgroundsthrough the air such that it performs a backward somersault whenreleased; the landing position is observed

2. Contact righting reflex—mouse is placed into a plastic tube andturned upside down

3. Negative geotaxis—mouse is placed no horizontal grid; the grid israised to vertical with mouse facing the floor; behavior is observed for30 seconds

The test results presented in FIGS. 7A-7C demonstrate that the compoundof Example 3 induced significant behavioral effects in the IrwinBehavioral Batter. FIG. 7A shows the results on spontaneous activity.FIG. 7B shows the results on grip strength. FIG. 7C shows the effects ofthe compound administration on limb tone.

Open Field testing was also performed. In carrying out this testing,individually housed male and female mice aged 8-12 weeks were used. Themice were maintained on a reverse L:D/12 p.m.: 12 a.m. cycle in abarrier facility. Food and water were available ad libitum. Testingoccurred towards the end of the light cycle between 6-12 a.m. Mice wereacclimated to the testing room for at least 30 minutes. The assay wasperformed in a custom-made Open Field Apparatus. Each chamber was a 50by 50 cm square. The experiment was recorded and tracked by a trackingsystem obtained from Viewpoint.

The time and the path length in the center of the open field weredetermined. The center of the open field is defined as a 13.5×13.5 cmsquare in the geometric center of the arena. The percent of path in thecenter is calculated as

$\frac{{Path}\mspace{14mu}{length}\mspace{14mu}{in}\mspace{14mu}{the}\mspace{14mu}{center}}{{Total}\mspace{14mu}{path}\mspace{14mu}{length}} \times 100\%$For each mouse, the total path length and path length for 60 minutes at5 minute intervals was determined as a measure of locomotor activity. Inaddition, feces produced by each experimental animal during the test wascounted. Each chamber was cleaned between individual mouse testing.

Path length for 60 minutes at 5 minute intervals was analyzed by repeatmeasures using SPSS software. All other parameters were compared usingunpaired t-test (GraphPad Prism).

The data presented in FIG. 8 demonstrates that the compound of Example 3is effective to reduce locomotor activity in open field testing. In yetanother assay, the same compound produced demonstrable anxiolyticeffects. See FIGS. 9A-9D.

Novel environment-induced feeding suppression (NEIFS) testing was alsoperformed. Individually housed male mice aged 8-12 weeks are used forthis testing. Mice were maintained no a reverse L:D/12 p.m.: 12 a.m.cycle in a barrier facility with food and water available ad libitum.Testing occurred towards the end of the light cycle between 6-12 a.m.The mice were acclimated to the testing room for at least 30 minutes.

Days 1, 2, 3 and 5 (Home cage): A Petri dish containing crushed Grahamcrackers was placed in the farthest corner from each mouse in its ownhome cage. The time to approach (defined as nose of the mouse directedat or within 1 cm of the dish) and consume (eat, not pick up) thecrackers was recorded immediately following the placement of the dishinto home cage.

Day 4 (Novel environment): The procedure followed was the same asdescribed for home cage; however, on day 4 the mouse was placed in a newcage with bedding (novel environment) for the duration of theexperiment. The mice were subsequently returned to their respective homecages for day 5 testing. The remaining crackers and Petri dish weredisposed of at the end of the experiment for each mouse.

In a novel environment induced feeding suppression assay, the compoundof Example 3 induced measurable anxiolytic effects. See FIG. 10.Notably, the compound had no effect on the number of fecal boli in openfield testing.

Rotarod assays were also performed using the experimental protocol anddesign set forth below. Individually housed male mice aged 8-12 weekswere used for this experiment. Mice were maintained on a reverse L:D/12p.m.: 12 a.m. cycle in a barrier facility with food and water availablead libitum. Testing occurred towards the end of the light cycle between6-12 a.m. Mice were acclimated to the testing room for at least 30minutes.

The assay was carried out using four EzRod test chambers kept in alaminar hood for testing. For the accelerating rotarod paradigm, micewere given 10 trials with the maximum duration of 3 minute and a30-second intertrial interral (ITI). Each mouse was placed on the EZRodmachines and the latency to fall was recorded for all trials. If themouse fell or 3 minutes elapsed, the mouse was left in the bottom of theEzRod test chamber for 30 seconds before starting the next trial.

The latency to fall was compared between the two groups by analysis ofvariance (ANOVA) with repeated measures.

The data presented in FIG. 11 reveal that the compound of Example 3 didnot affect rotarod performance.

The foregoing specification includes citations to certain patent andliterature references, which are provided to indicate the state of theart to which this invention pertains. The entire disclosure of each ofthe cited references is incorporated by reference herein.

While certain embodiments of the present invention have been describedand/or exemplified above, various other embodiments will be apparent tothose skilled in the art from the foregoing disclosure. The presentinvention is, therefore, not limited to the particular embodimentsdescribed and/or exemplified, but is capable of considerable variationand modification without departure from the scope of the appendedclaims.

What is claimed is:
 1. The compound3-(phenylpropyl)-sydnonimine-N-phenylcarbamoyl and the pharmaceuticallyacceptable salts of said compound.
 2. A pharmaceutical compositioncomprising a compound according to claim 1 and a pharmaceuticallyacceptable carrier medium.
 3. A composition according to claim 2 insolid form, also comprising a pharmaceutically acceptable excipient. 4.A composition according to claim 2 in liquid form, also comprising apharmaceutically acceptable diluent.
 5. A composition according to claim2 in unit dosage form.
 6. A composition according to claim 2, furthercomprising at least one selective serotonin reuptake inhibitor.